The present invention relates to novel drugs for use in combating, for example, infectious microorganisms, particularly bacteria. More specifically, the invention relates to peptide nucleic acid (PNA) sequences that are modified in order to obtain novel PNA molecules which exhibit enhanced anti-infective properties.
The discovery of penicillin in the 1940""s marked the beginning of the search for new antibiotics. Many antibiotics have been discovered or developed from existing drugs, and the number of antibiotic drugs currently used by clinicians is more than 100. Many strains of bacteria have, unfortunately, become resistant to one or more of the currently available antibiotics.
Most antibiotics are products of natural microbic populations and resistant traits found in these populations can disseminate between species and appear to have been acquired by pathogens under selective pressure from antibiotics used in agriculture and medicine (Davis et al., Science, 1994, 264, 375). Antibiotic resistance may develop in bacteria harbouring genes that encode enzymes that either chemically alter or degrade antibiotics. Resistant bacteria may also encode enzymes that make the cell wall impervious to antibiotics or encode efflux pumps that eject antibiotics from the cell before the antibiotics can exert their effects. Due to the emergence of antibiotic-resistant bacterial pathogens, a need for new therapeutic strategies has arisen. One strategy for avoiding problems caused by resistance genes is the development of anti-infective drugs from novel chemical classes for which specific resistance traits do not exist.
Antisense agents offer a novel strategy for combatting disease, as well as opportunities to employ new chemical classes in drug design. Oligonucleotides can interact with native DNA and RNA in several ways, including duplex formation between an oligonucleotide and a single-stranded nucleic acid and triplex formation between an oligonucleotide and double-stranded DNA to form a triplex structure. The use of anti-sense methods in basic research has been encouraging, and antisense oligonucleotide drug formulations that target viral and disease-causing human genes are progressing through clinical trials. Antisense inhibition of bacterial genes could also have wide application; however, few attempts have been made to extend antisense technology to bacteria.
Peptide nucleic acids (PNA) are similar to oligonucleotides and oligonucleotide analogs and may mimic DNA and RNA. The deoxyribose backbone of DNA is replaced in PNA by a pseudo-peptide backbone (Nielsen et al., Science, 1991, 254, 1475; see FIG. 1). Each subunit, or monomer, has a naturally occurring or non-naturally occurring nucleobase attached to the backbone. One such backbone consists of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. PNA hybridizes to complementary nucleic acids through Watson and Crick base pairing and helix formation results (Egholm et al., Nature, 1993, 365, 566). The Pseudo-peptide backbone provides superior hybridization properties (Egholm et al., Nature, 1993, 365, 566), resistance to enzymatic degradation (Demidov et al., P.E. Biochem. Pharmacol., 1994, 48, 1310) and access to a variety of chemical modifications (Nielsen et al., Chemical Society Reviews, 1997, 73).
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes, as determined by Tms. The thermal stability of PNA/DNA and PNA/RNA duplexes could be due to the lack of charge repulsion in the neutral backbone of PNA. In addition to increased affinity, PNA has also been shown to hybridize to DNA with increased specificity, as compared to DNA/DNA duplexes. When a PNA/DNA duplex mismatch is melted relative to a DNA/DNA duplex, an 8 to 20xc2x0 C. drop in the Tm results. Furthermore, homopyrimidine PNA oligomers form extremely stable PNA2-DNA triplexes with sequence-complementary targets in DNA or RNA oligomers. Finally, PNAs may bind to double-stranded DNA or RNA by helix invasion.
One advantage of PNA, as compared to oligonucleotides, is the nuclease and protease reisitance of the PNA polyamide backbone. PNA is not recognized by either nucleases or proteases and is thus not susceptible to cleavage; consequently, PNAs are resistant to degradation by enzymes, unlike nucleic acids and peptides. In antisense applications, target-bound PNA can cause steric hindrance of DNA and RNA polymerases, reverse transcripase, telomerase and ribosomes (Hanvey et al., Science, 1992, 258, 1481; Knudsen et al., Nucleic Acids Res., 1996, 24, 494; Good at el., Proc. Natl. Acad. Sci USA, 1998, 95, 2073; Good, et al., Nature Biotechnology, 1998, 16, 355).
A general difficulty in the use of antisense agents is cell uptake. A variety of strategies to improve uptake have been explored and reports of improved uptake into eukaryotic cells using lipids (Lewis et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 3176), encapsulation (Meyer et al., J Biol. Chem., 1998, 273, 15621) and carrier strategies (Nyce et al., Nature, 1997, 385, 721; Pooga et al., Nature Biotechnology, 1998, 16, 857) have been made. WO 99/05302 discloses a PNA conjugate consisting of PNA and the transporter peptide transportan, in which the peptide can be used for transport cross a lipid membrane and for delivery of the PNA into interactive contact with intracellular polynucleotides. U.S. Pat. No. 5,777,078 discloses a pore-forming compound which comprises a delivery agent that recognizes the target cell and is linked to a pore-forming agent, such as a bacterial exotoxin. The compound is administered together with a drug such as PNA.
PNAs have unique advantages as an antisense agent for microorganisms. PNA-based antisense agents can control cell growth and growth phenotypes when targeted to Escherichia coli rRNA and mRNA (Good et al., Proc. Natl. Acad. Sci USA, 1998, 95, 2073; Good et al., Nature Biotechnology, 1998, 16, 355; and WO 99/13893).
None of the cited disclosures discuss methods of transporting PNA across the bacterial cell wall and membrane. Poor uptake of PNA is expected because bacteria have stringent barriers against entry of foreign molecules and antisense oligomer-containing nucleobases appear to be too large for efficient uptake. The results obtained by Good and Nielsen (Good et al., Proc. Natl. Acad. Sci USA, 1998, 95, 2073; Good, et al., Nature Biotechnology, 1998, 16, 355) indicate that PNA oligomers enter bacterial cells poorly by passive diffusion across the lipid bilayers.
U.S. Pat. No. 5,834,430 discloses the use of potentiating agents, such as short cationic peptides, in the potentiation of antibiotics. The agent and the antibiotic are co-administered. WO 96/11205 discloses PNA conjugates, wherein a conjugated moiety may be placed on terminal or non-terminal parts of the backbone of PNA in order to functionalize the PNA. The conjugated moieties may be reporter enzymes or molecules, steroids, carbohydrate, terpenes, peptides, proteins, etc. The conjugates possess improved transfer properties for crossing cellular membranes; however, WO 96/11205 does not disclose conjugates that can cross bacterial membranes.
WO 98/52614 discloses a method of enhancing transport over biological membranes, e.g., a bacterial cell wall. According to this publication, biologically active agents such as PNA may be conjugated to a transporter polymer in order to enhance transmembrane transport. The transporter polymer consists of 6-25 subunits, at least 50% of which contain a guanidino or amidino side chain moiety, and wherein at least 6 contiguous subunits contain guanidino and/or amidino side chains. A preferred transporter polymer is a polypeptide containing 9-arginine. Despite the promising results obtained with the use of the PNA technology, there is a great need in the art for development of new PNA antisense drugs that are effective in combatting microorganisms.
The present invention relates to a new strategy for combatting bacteria. Antisense PNA can inhibit the growth of bacteria; however, due to slow diffusion of PNA across the bacterial cell wall, the use of PNA as an antibiotic has not been possible. According to the present invention, a practical application for PNA in combatting bacteria can be achieved by modifying the PNA through linkage of a peptide or peptide-like sequence that enhances the activity of the PNA.
Surprisingly, it has been demonstrated that incorporation of a peptide in PNA results in an enhanced anti-infective effect. An important feature of the modified PNA molecules is a pattern comprising positively charged and lipophilic amino acids or amino acid analogues. An anti-infective effect is found with different orientations of the peptide relative to the PNA sequence. Thus, one aspect of the present invention is directed to a modified PNA molecule, and pharmaceutically acceptable salt thereof, of Formula I:
xe2x80x83Peptide-L-PNAxe2x80x83xe2x80x83(I)
wherein L is a linker or a bond, Peptide is any amino acid sequence, and PNA is a Peptide Nucleic Acid.
More particularly, the present invention is directed to a modified PNA molecule of Formula I
Peptide-L-PNAxe2x80x83xe2x80x83(I)
wherein Peptide is a cationic peptide or cationic peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the Formula II:
C-(B-A)n-D, xe2x80x83xe2x80x83(II)
wherein A is from 1 to 8 non-charged amino acids and/or amino acid analogs, B is from 1 to 3 positively charged amino acids and/or amino acid analogs, C is from 0 to 4 non-charged amino acids and/or amino acid analogs, D is from 0 to 3 positively charged amino acids and/or amino acid analogs, n is 1-10, and the total number of amino acids and/or amino acid analogs is from 3 to 20.
In one embodiment, the Peptide of the present invention comprises from 2 to 60 amino acids. The amino acids can be negatively charged, non-charged, or positively charged naturally-occurring, rearranged, or modified amino acids. In a preferred embodiment of the invention, the peptide comprises from 2 to 18 amino acids, and most preferably from 5 to 15 amino acids.
In another preferred embodiment of the invention, A in Formula II comprises from 1 to 6 non-charged amino acids and/or amino acid analogs and B comprises 1 or 2 positively charged amino acids and/or amino acid analogs. In another embodiment, A comprises from 1 to 4 non-charged amino acids and/or amino acid analogs and B comprises 1 or 2 positively charged amino acids and/or amino acid analogs.
In a preferred embodiment of the invention, the modified PNA molecules of Formula I are used, for example, in the treatment or prevention of infections caused by Escherichia coli or vancomycin-resistant enterococci such as Enterococcus faecalis and Enterococcus faecium or infections caused by methicillin-resistant and methicillin-vancomycin-resistant Staphylococcus aureus. 
The peptide moiety in a modified PNA molecule is linked to the PNA sequence via the amino (N-terminal) or carboxy (C-terminal) end. In a preferred embodiment, the peptide is linked to the PNA sequence via the carboxy end.
In another aspect of the invention, modified PNA molecules are used in the manufacture of medicaments for the treatment or prevention of infectious diseases or for disinfecting non-living objects. In a further aspect, the invention concerns a composition for treating or preventing infectious diseases or disinfecting non-living objects. In yet another aspect, the invention concerns the treatment or prevention of infectious diseases or treatment of non-living objects.
In yet a further aspect, the present invention is directed to a method of identifying specific advantageous antisense PNA sequences that may be used in the modified PNA molecule according to the invention.
In yet a further aspect, the present invention relates to other antisense oligonucleotides with the ability to bind to both DNA and RNA.
Oligonucleotide analogs are oligomers having a sequence of nucleotide bases (nucleobases) and a subunit-to-subunit backbone that allows the oligomer to hybridize to a target sequence in an mRNA by Watson-Crick base pairing, to form an RNA/Oligomer duplex in the target sequence. The oligonucleotide analog may have exact sequence complementarity to the target sequence or near complementarity, as long as the hybridized duplex structure formed has sufficient stability to block or inhibit translation of the mRNA containing target sequence.
Oligonucleotide analogs of the present invention are selected from the group consisting of Locked Nucleoside Analogues (LNA) as described in International PCT Publication WO99/14226, oligonucleotides as described in International PCT Publication WO98/03533 or antisense oligomers, in particular morpholino analogs as described in International PCT Publication W098/32467.
PCT Publication WO99/14226, WO98/03533 and WO98/32467 are all incorporated by reference.
Thus, further preferred compounds of the invention are modified oligonucleotides of the Formula (III):
Peptide-L-Oligonxe2x80x83xe2x80x83(III)
wherein L is a linker or a bond; Peptide is any amino acid sequence and Oligon is an oligonucleotide or analog thereof.